Cryptic splicing events in the iron transporter ABCB7 and other key target genes in SF3B1-mutant myelodysplastic syndromes.

H Dolatshad,S Roy,F G Liberante,C W J Smith,S Killick,B H Yip,R N Armstrong,M Llorian,K I Mills,R Kušec,E Repapi,L Scifo,V Steeples,J Shaw,S Taylor,K I Savage,J Boultwood,A Pellagatti

doi:10.1038/leu.2016.149

Abstract

The splicing factor SF3B1 is the most frequently mutated gene in myelodysplastic syndromes (MDS), and is strongly associated with the presence of ring sideroblasts (RS). We have performed a systematic analysis of cryptic splicing abnormalities from RNA sequencing data on hematopoietic stem cells (HSCs) of SF3B1-mutant MDS cases with RS. Aberrant splicing events in many downstream target genes were identified and cryptic 3′ splice site usage was a frequent event in SF3B1-mutant MDS. The iron transporter ABCB7 is a well-recognized candidate gene showing marked downregulation in MDS with RS. Our analysis unveiled aberrant ABCB7 splicing, due to usage of an alternative 3′ splice site in MDS patient samples, giving rise to a premature termination codon in the ABCB7 mRNA. Treatment of cultured SF3B1-mutant MDS erythroblasts and a CRISPR/Cas9-generated SF3B1-mutant cell line with the nonsense-mediated decay (NMD) inhibitor cycloheximide showed that the aberrantly spliced ABCB7 transcript is targeted by NMD. We describe cryptic splicing events in the HSCs of SF3B1-mutant MDS, and our data support a model in which NMD-induced downregulation of the iron exporter ABCB7 mRNA transcript resulting from aberrant splicing caused by mutant SF3B1 underlies the increased mitochondrial iron accumulation found in MDS patients with RS.

Highlights

The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis leading to peripheral blood cytopenias, and show increasing bone marrow blasts.[1]The MDS show frequent progression to acute myeloid leukemia
Samples and RNA sequencing (RNA-Seq) RNA-Seq data were obtained from CD34+ cells isolated from bone marrow samples of eight MDS patients with SF3B1 mutation, four MDS cases without mutations in the splicing factor genes SF3B1, SRSF2, U2AF1 and ZRSR2, and five healthy individuals.[20]
Cryptic splicing events in HSCs of SF3B1-mutant MDS We have analyzed RNA-Seq data obtained from the CD34+ cells from eight MDS cases harboring SF3B1 mutations (SF3B1-mutant, all with 415% ring sideroblasts (RS)), four MDS patients without splicing factor gene mutations and five healthy individuals[20] using rMATS, a bioinformatics pipeline designed to detect alternative splicing events involving two isoforms from an alternatively spliced region.[32]

Summary

Introduction

The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis leading to peripheral blood cytopenias, and show increasing bone marrow blasts.[1]. The MDS show frequent progression (approximately 40% of patients) to acute myeloid leukemia. Several genes involved in pre-messenger RNA splicing, including SF3B1, U2AF1, SRSF2 and ZRSR2,2–4 have been shown to be mutated in over 50% of MDS patients, revealing a new leukemogenic pathway involving spliceosomal dysfunction. The clinical consequences of mutations in SF3B1 are well documented in MDS, the functional consequences of SF3B1 mutations in human hematopoietic cells are not fully understood

Methods

Results

Conclusion